Project Summary/Abstract Circadian clocks influence nearly all aspects of mammalian life, aligning our internal physiological process to optimal times of day. Understanding the molecular circuitry keeping circadian time provides insight into how the clock drives overt rhythms, and what to fix when the circadian system is disrupted (i.e. during shift work). Circadian time coded in the rhythmic regulation of ?clock gene? expression in a negative feedback loop system. Critical to this timing system is the circadian degradation of rhythmically abundant clock proteins, however these mechanisms have remained elusive. We have begun elucidating these mechanisms by developing a novel functional screening approach designed to identify which E3 ubiquitin ligases degrade which clock proteins. Screen data on three clockwork targets indicate this approach is sensitive and specific, thus operating as we hoped. A major goal of the current application is to continue to screen for, and validate, E3 ligases for other clockwork proteins as well as some key rhythmic circadian outputs. We believe identifying these E3 ligases and their roles in clock function will reveals new potential targets for therapeutic intervention of clock- related disorders. The first screen hit we have recovered, Siah2, has revealed remarkable and unexpected new insights into both clock function and how the clock regulates metabolism. We found that female mice lacking a functional Siah2 gene lost a mechanism that ?protects? against diet- induced obesity. This appears to be due to a female-specific role in the core circadian clock. These results suggest, for the first time, that female-specific clockwork mechanisms exist, and that they contribute directly to female-specific biology. Unfortunately, the vast majority of circadian data are from male mice. Therefore, we now plan to reassess clock function and circadian rhythm regulation in females to further identify and define the sex-specific clockwork mechanisms to provide a background for examining the role of Siah2 in female clocks. Understanding sex-differences in circadian clock functions will be critical as the field develops circadian-based therapeutics. !